Introduction

Histone modifications are central in regulating basic processes including transcription, gene silencing, differentiation, cell-cycle progression, and DNA repair (1–4). Among modifications of specific histone residues, much attention has focused in the last decade on a distinctive pathway, mediated by the E3 ligase Bre1, which monoubiquitinates histone H2B at lysine 123 (H2BK123) in budding yeast and at lysine 120 (H2BK120) in humans. H2B monoubiquitination plays an important role in regulation of transcription, being a prerequisite for normal levels of methylation of histone H3 residues K4 and K79 (5–8). In addition, studies in Saccharomyces showed that mutants deficient in H2B monoubiquitination are radiation sensitive and defective in recombinational repair, cell-cycle checkpoints, gene silencing, and meiosis (see ref. 9 for review). Several recent publications on the mammalian homolog of Bre1 (RNF20/RNF40) suggest that mammalian Bre1 complex plays similar roles. We have reported that loss of Bre1 in mouse cells compromised homologous recombination (HR) repair, resulting in reduced recruitment of Rad51 to sites of radiation-induced double-strand breaks (DSB) and increased sensitivity to ionizing radiation and DNA cross-linking agents (10). A subsequent study by others concluded that human Bre1 (RNF20, or BRE1A) was involved in chromatin reorganization, facilitating HR protein access to damaged DNA (11). This role of Bre1 seems to be important for response to DSBs in general, as a recent study has shown that BRE1 (RNF20/40)-mediated H2B ubiquitination is induced in an ATM-dependent manner and is essential for timely repair of DSBs (12). These findings place Bre1 at the forefront of the DNA damage response, which serves as a barrier against cancer formation (13), and reduction in Bre1 levels would be expected to compromise stability of the genome.

Given that Bre1 affects multiple functions involved in genome maintenance, it is plausible that Bre1 homologs have a major tumor-suppressing role in higher organisms. Consistent with this, the RNF20 promoter has been found to be hypermethylated in breast cancers (14). Shema and colleagues (14) showed that BRE1A restrained transcription of several proto-oncogenes, including c-myc and c-Fos, and augmented expression of tumor suppressor genes, notably p53, and further concluded that selective transcriptional regulation of proto-oncogenes and tumor suppressors constituted the basis for the tumor-suppressing activity of BRE1 (RNF20). According to this hypothesis, one consequence of the loss of Bre1 would be an increased proliferation resulting from upregulation of growth-promoting oncogenes. However, this conclusion seems to be at odds with our current observation of impaired growth upon depletion of Bre1 (RNF20/40). Also, the transcriptional regulation of select oncogenes and tumor suppressors does not seem to be universal across species, as in our study we detect no reduction of p53 mRNA levels after knockdown of Bre1 (Rnf20 and/or Rnf40) in mouse cells. Thus, additional mechanisms seem to be at play in Bre1-depleted cells driving their transition to a malignant phenotype.

Here, we show that Bre1 acts as an important suppressor of genomic instability. We used RNA interference (RNAi) to deplete Bre1 from mouse cells and then followed the evolution of genomic instability in Bre1-deficient cells from early replication stress to specific genomic rearrangements, which in turn triggered breakage–fusion–bridge cycles, known to accelerate genomic instability. We show that R-loops, the RNA–DNA hybrids that result from the extended pairing of nascent mRNA with the DNA template behind the elongating RNA polymerase II, are a significant contributor to the genomic instability phenotype in the Bre1-depleted cells. Although Bre1 homologs are highly expressed in mouse and human testis, we find reduced BRE1A and BRE1B levels in testicular cancer and the nonmalignant lesion in situ carcinoma. Collectively, our data show that Bre1 deficiency promotes genomic instability, which may be an early step in carcinogenesis preceding the acquisition of an invasive phenotype.

RNA interference

For downregulation of Bre1a and Bre1b by RNAi, we used a lentiviral RNAi system based on the BLOCK-iT system (Invitrogen) modified by Dr. E. Campeau (17).

Quantitative RT-PCR

Quantitative real-time PCR (qRT-PCR) analysis was done as described in Razorenova and colleagues (18). Human or mouse TBP was used for normalization of cDNA input. The amplification was specific as judged by melting temperature analysis. The experiments were conducted in triplicate and repeated at least twice. Primer sequences are available upon request.

Visualization of micronuclei and anaphase bridges

The assay was done as described in Chernikova and colleagues (19). In some cases (Fig. 2B, Supplementary Fig. S3B and S3C), cytochalasin B was not added to allow better visualization of anaphase bridges.

Rad51 foci detection

RNase H1 transfection

GFP-RNase H1 plasmid was obtained from Dr. Olivier Sordet (Cancer Research Center of Toulouse, France), and transfection carried out using procedures similar to described in Sordet and colleagues (20) and in Supplementary Information.

More detailed Materials and Methods can be found in Supplementary Information.

Results

Bre1a/Bre1b-depletion impairs cell growth

To investigate the effects of loss of function of the Bre1a/Bre1b complex in mouse cells, we created lentivirus-based short hairpin RNA (shRNA) constructs targeting either of the 2 subunits of the complex. As a control, we used an shRNA construct targeting GFP. Expression of Bre1a and Bre1b shRNAs in mouse fibrosarcoma RIF-1 cells resulted in a significant reduction of corresponding mRNA transcript levels after 7 days of antibiotic selection, as shown by qRT-PCR; Fig. 1A). Protein levels of Bre1a and Bre1b were also reduced (Fig. 1B). Importantly, the levels of Bre1b protein were decreased in the knockdown of Bre1a (Fig. 1B, top panel), whereas the levels of Bre1b mRNA were not affected by the knockdown of Bre1a (Fig. 1A), suggesting that stability of Bre1b depends on the formation of Bre1a/Bre1b heterodimer.

Depletion of Bre1a or Bre1b impairs cell growth. A, qRT-PCR showing reduced mRNA levels of Bre1a or Bre1b in the knockdowns of Bre1a or Bre1b, respectively. B, knockdown of Bre1a or Bre1b results in decrease in H3K4me3 and H3K79me2. C, depletion of Bre1a or Bre1b decreases monoubiquitination of histone H2B. D, depletion of Bre1a and Bre1b results in slow growth of RIF-1 cells. E, conditional expression of Bre1b shRNA in mouse RIF-1 cells results in slow growth (a), increased apoptosis (as measured by Annexin–FITC staining; b), and increased induction of micronuclei (c). Error bars are SEM. Experiments were repeated more than 3 times. (+) and (−) show presence or absence of doxycycline in the medium. F, representative microphotograph showing micronuclei in RIF-1 cells after knockdown of Bre1a (panel a) or Bre1b (panel b).

In agreement with previous studies (21), we showed that downregulation of either Bre1a or Bre1b leads to reduction in ubiquitination of H2B (Fig. 1C) and subsequent reduction of uH2B-dependent methylation of H3K79 and H3K4 (Fig. 1B). The reduction in H3K4me3 was evident in all knockdowns but was substantially less pronounced than the reduction in H3K79me2 (Fig. 1B), which closely followed the reduction in Bre1a or Bre1b levels in all experiments.

Knockdown of Bre1a or Bre1b significantly impaired cell growth in different mouse cell types including RIF-1, C3H 10T1/2, and MES cells (Fig. 1D and Supplementary Fig. S1A), whereas cells grew well after knockdown of either GFP, or tumor suppressor Rb (Supplementary Fig. S1A). To monitor the effects of disruption of the Bre1a/Bre1b complex over time, we created RIF-1–derived cell lines, in which the knockdown of Bre1a or Bre1b could be initiated by addition of doxycycline. A doxycycline-inducible GFP shRNA system was used as a control. A detailed description of the Bre1 shRNA-inducible system is presented in the Supplementary Materials and Methods and in Supplementary Fig. S1B and S1C. Note that the Bre1b shRNA–inducible cell line has a slightly reduced level of Bre1b, even in the absence of doxycycline, compared with the control cell line with GFP shRNA (Supplementary Fig. S1C, lanes 1 and 4, respectively), indicating that the doxycycline-inducible system is leaky and a partial knockdown of Bre1b occurs even when doxycycline is not present. Similarly to cells with the constitutive Bre1b knockdown (Fig. 1B), the inducible Bre1b shRNA cell line showed significant reduction in growth rate upon addition of doxycycline (Fig. 1E, panel a). Consistent with the leaky phenotype, the growth rate of the inducible Bre1b shRNA cells was slightly reduced even in the absence of doxycycline, when compared with the control-inducible GFP shRNA cell line (Fig. 1E). We were able to obtain a more tightly regulated cell line by modifying stringencies of antibiotics' selection (shBre1b2 in Fig. 2A), which corrected the problem of reduced cell growth in the absence of doxycycline. The availability of the leaky cell line shBre1b1 with partial knockdown of Bre1b gave us an advantage to investigate weaker and/or long-term effects of Bre1b depletion (shown further).

Evidence for ongoing genomic instability in Bre1-depleted cells. A and B, Bre1b depletion leads to increased frequency of micronucleus (MN) formation. Note different levels of Bre1b, uH2B, and H3K79me2 in 2 doxycycline-inducible cell lines, shBre1b1 and shBre12. shBre1b1, a Bre1b shRNA doxycycline-inducible cell line shows a partial Bre1b knockdown even in the absence of doxycycline. shBre1b2 is derived from early passage shBre1b1 cells line by increasing the stringency of blasticidin selection (from 5 to 10 μg/mL). The (+) and (−) show presence or absence of doxycycline in the medium. C, anaphase bridges in Bre1b-depleted cells contain telomeric sequences (arrowhead). D, Bre1b-depleted cells are characterized by appearance of aberrations with amplified centromeric heterochromatin (stained by 4′, 6-diamidino-2-phenylindole). Shown is a RTCH. E, long-term partial knockdown of Bre1b in the shBre1b1 cell line leads to multiple chromosomal abnormalities, which increase in frequency and complexity. Abbreviations: CA, chromatid-type aberrations; RT, classic Robertsonian translocations; ACH, complex aberrations involving multiple amplification of heterochromatin. Note that RTCH frequencies are much higher in shBre1b1 cells at later passages. Significance analysis is provided in Supplementary Table S5. F, representative examples of observed chromosome aberrations: panels a and b–chromatid-type (CA) aberrations, panels c, d and e—complex aberrations involving heterochromatin (ACH). Arrowhead points to RTCH in (F) panel e.

Apoptosis contributes to growth impairment in Bre1-depleted cells

To explain the reduction in growth rate observed in the Bre1-deficient cells, we investigated the possibility of increased apoptosis. Apoptosis frequencies after Bre1b knockdown were increased by up to 5-fold compared with uninduced cells, and to both induced control and uninduced control cells (Fig. 1E (panel b) and Supplementary Fig. S2A). Consistent with the leaky phenotype, low level of apoptosis was present in the shBre1b1 cells even in the absence of doxycycline (Fig. 1E, panel b). Apoptosis also increased after depletion of Bre1 in MES cells (Supplementary Fig. S2B). We concluded that the function of Bre1 is critical for homeostasis and that apoptosis contributes to the reduced growth phenotype in cell cultures with low levels of Bre1a and Bre1b.

Bre1 loss compromises genomic integrity

Reduction in growth rate and increased apoptosis also closely correlated with increased frequencies of micronuclei (MN; Fig. 1E and F), supporting the conclusion that the function of the Bre1a/Bre1b complex is essential for maintenance of DNA integrity. Importantly, we observed a tight correlation between H2B ubiquitination and MN levels (Supplementary Fig. S3A and S3B).

Progressive genomic instability in Bre1-deficient cells

Although significant loss of Bre1 was catastrophic to the cells and lead to cessation of proliferation and cell death (Fig. 1E), more subtle changes in Bre1b expression due to the partial knockdown in the uninduced shBre1b1 cell line allowed cells to proliferate and manifest dramatic genomic instability associated with Bre1 deficiency after longer culture periods. When cultured for 3 weeks and longer under conditions of partial knockdown (Fig. 2A), the shBre1b1 cells displayed MN frequencies comparable with that observed on day 5 after complete Bre1b knockdown in the shBre1b2 cells (Fig. 2B). In addition, partial knockdown shBre1b1 cells at higher passages were characterized by the appearance of anaphase bridges (Fig. 2C and Supplementary Fig. S3C), many of which contained telomere signal(s) (Fig. 2C), and by an increased DNA content (Supplementary Fig. S4). To differentiate between fusions resulting from loss of telomere end protection (22, 23) and fusions resulting from DSBs (24, 25), we carried out telomere FISH analysis on metaphase chromosomes from the Bre1-deficient and control cell lines. Because no telomere signal was observed interstitially at fusion points, chromosome analysis provided no evidence for the loss of telomere end capping function, suggesting that anaphase bridges resulted from DSBs.

We also found that loss of Bre1 led to the formation of 3 main types of chromosome aberrations, providing insight into mechanisms underlying the formation of telomere-positive anaphase bridges and to the increased DNA content in the Bre1-knockdown cells. The chromosome aberrations predominantly found in the Bre1-knockdown cells were Robertsonian-like translocations with amplified centromeric heterochromatin (RTCH; Fig. 2D), aberrations involving large regions of amplified centromeric heterochromatin at pericentric and interstitial locations (ACH), and chromatid-type aberrations (CA; Fig. 2E and F), aberrations associated with instability. The amplified regions of centromeric heterochromatin observed in the metaphase chromosomes of the Bre1-knockdown cell line were sometimes involved in dicentric chromosomes, which lead to the generation of anaphase bridges. These bridges contained telomere signals not associated with any telomere end protection defect and subsequent chromosomal end fusions, but rather telomeres associated with chromosomes pulled into the nucleoplasmic tube of an anaphase bridge, providing explanation for the presence of telomere signals within the anaphase bridges of the Bre1-knockdown cell line.

The chromosomal instability phenotype initiated by partial Bre1b knockdown (shBre1b1 cells grown without doxycycline) for 3 weeks and longer was more dramatic than the one initiated by complete short-term knockdown of Bre1b at early passage (Fig. 2E and F). In addition, cells at higher passages accumulated chromatid-type aberrations, which were not present in the lower passage cells, as well as very complex aberrations, which seemed to result from disruption/amplification/decompaction of centromeric heterochromatin (Fig. 2E and F).

Bre1 deficiency leads to replication stress

To elucidate how loss of Bre1 contributes to increased DSBs and genomic rearrangements, we measured γH2AX levels after Bre1 knockdown. Consistent with previous reports (26, 27), we found 2 distinct subpopulations of cells with elevated γH2AX. In the first, an increase in γH2AX signal was detected as a shift of the main cell population in the fluorescence-activated cell sorting (FACS) profile (Fig. 3A) that could be accounted for by both an increase in the intensity of γH2AX foci and/or in the number of cells with γH2AX foci at sites of DSBs (Fig. 3B–D panels a and b). The other population of cells had dramatically elevated pan-nuclear staining of γH2AX (Fig. 3D panel c), which identified apoptotic or preapoptotic cells (26, 27). FACS analysis showed that although γH2AX signal (DSBs) was elevated throughout the cell cycle (Fig. 3A), cells with pan-nuclear staining of γH2AX (apoptosis) were restricted to S-phase cells after knockdown of Bre1 (Fig. 3E). To investigate how Bre1-deficient cells dealt with replication stress, we treated cells with 2 mmol/L hydroxyurea (HU), which stalls replication forks by inhibiting ribonucleotide reductase essential for the synthesis of DNA precursors. A prolonged (24 hours) treatment with 2 mmol/L HU leads to replication fork collapse, which requires HR for repair of resulting DSBs (28). We found that prolonged treatment of control cells with 2 mmol/L HU led to a distribution of γH2AX signal reported previously (29), with γH2AX-positive cells in 3 distinct clusters corresponding to G1, S, and G2-M cells (Fig. 3F). Consistent with increased apoptosis during replication, we also found that loss of Bre1 resulted in depletion of cells primarily from the cluster corresponding to the γH2AX-positive S-phase cells (Fig. 3F). Replication stress and occurrence of chromatid aberrations are consistent with defective homologous recombination in Bre1-depleted cells we reported previously, and we showed that spontaneous Rad51 foci formation was significantly affected by loss of Bre1 (Supplementary Fig. S4C).

Bre1 knockdown leads to increase in DSBs and to replication stress. A, FACS analysis shows that loss of Bre1b elevates γH2AX. A representative experiment is shown. Increase in γH2AX signal results from an increase in intensity of γH2AX foci (B) and from increased number of cells counted as γH2AX-positive (C; ***, P < 0.001, t test). D, panel a shows cells with low γH2AX signal and panels b and c show cells with more intense γH2AX foci after Bre1 knockdown. Panel c shows a preapoptotic cell with very bright γH2AX signal. DAPI, 4′, 6-diamidino-2-phenylindole. E, Bre1-deficient cells show more cells with high γH2AX signal specifically in S-phase. For quantitation purposes FACS settings were adjusted so that the cells were in the same location on the plots. Note that in the corresponding Western blot % of γH2AX-positive cells is inversely proportional to the levels of uH2B in the Bre1a or Bre1b knockdowns. F, twenty-four-hour treatment with hydroxyurea leads to different distribution of γH2AX signal in the Bre1 knockdown cells. (+) and (−) show presence or absence of doxycycline in medium. Experiments were repeated more than 3 times.

To explore additional factors contributing to DSB formation in Bre1-deficient cells, we carried out gene expression analysis on Bre1a and Bre1b knockdown cells. Because growth-affecting changes were observed after longer times of Bre1 knockdown (Fig. 1D), we carried out microarray analysis after 7 days of RNAi. Consistent with both subunits of Bre1 complex being required for the H2B ubiquitination, we observed a high correlation between expression profiles in the Bre1a and Bre1b knockdowns (Supplementary Fig. S5). Despite the general role of Bre1 in transcriptional regulation, loss of Bre1 downregulated a distinct set of genes and upregulated another without affecting majority of genes (Supplementary Tables S1 and S2). Further analysis revealed that the group of genes upregulated after Bre1 knockdown was dramatically enriched for genes involved in RNA processing/splicing, showing a response typical of the one to cotranscriptional formation of recombinogenic RNA–DNA hybrids (R-loops; Fig. 4A). R-loops often arise during perturbed mRNA processing as a result of the extended pairing of nascent mRNA with the transcribed DNA strand behind the elongating Pol II, creating DSBs and leading to increased recombination and genomic instability (30, 31). In a genome-wide siRNA screen for genes whose deregulation leads to elevated levels of γH2AX (32), the mRNA processing module represented the most significantly enriched category of genes, suggesting that abnormal mRNA processing was the most common and direct source of genomic instability. To determine whether R-loop formation contributed to increased formation of DSBs in Bre1-deficient cells, we tested whether overexpression of RNase H1, which degrades RNA in R-loops, would reduce levels of γ-H2AX. Figure 4B shows that overexpression of RNase H1 decreased the number of cells with γ-H2AX foci in the Bre1-depleted cells, whereas it did not have an effect on cells in which Bre1 RNAi was not induced. In addition, RNase H1 expression reduced the average number of γ-H2AX foci per cell (Fig. 4C and D). These data suggested a mechanism for the generation of DSBs through the R-loop formation in the Bre1-depleted cells.

Defect in mRNA processing contributes to DSB formation in Bre1-depleted cells. A, classification enrichment in gene sets downregulated and upregulated after Bre1 knockdown. Pathway/process enrichment analysis was done using DAVID bioinformatics database (Supplementary Methods). The statistical threshold was applied for −log(P = 0.05) shown in dashed line. B, overexpression of RNase H1 in the shBre1b cells results in reduction of cells with γH2AX foci. C, overexpression of RNase H1 in Bre1b-depleted cells reduces number of γH2AX foci per cell. D, a representative photograph of an experiment from (C). The cells were transfected with GFP-RNase H1 or GFP-Nuc (control) on day 3 after the induction of knockdown and were analyzed on day 6 after the induction of knockdown. In (B) the GFP-expressing cells were FACS sorted on day 5 after the induction of knockdown and plated for analysis to be carried out next day. The FACS sorting experiments were conducted twice. In (C) cells transfected with GFP-RNase H1 were analyzed without prior sorting. Asterisks denote a significant difference from cells with Bre1 depletion, but not expressing RNase H1 (***, P < 0.001, t test).

We also found that the gene subset upregulated after depletion of Bre1 was strongly enriched for histone genes from the compact chromatin cluster on 13qA3.1 (P < 0.0001; Supplementary Table S3). Normally, replication-dependent histone mRNAs do not have poly(A) tails, but as we relied on poly(A)-dependent amplification of mRNA for hybridization to our microarray chip, overrepresentation of histone genes from the 13qA3.1 cluster is a compelling evidence for the presence of mRNA that has been polyadenylated. These data confirmed the previous finding by Pirngruber and colleagues (33) showing that BRE1(RNF20/40)-dependent H2Bub1 acts as a marker for correct recognition of the histone mRNA 3′-end cleavage site. Due to the massive synthesis of histones during replication, defective processing of replication-associated histone mRNA in BRE1(RNF20/40)-deficient cells may be the main contributor to the formation of DSBs and hence replication stress.

BRE1A/B and H3K79me2 levels are lower in human seminoma than in normal testis

We hypothesized that tissues with high expression of Bre1 homologs would depend on the function of the complex for maintenance of genomic integrity. Western blot analysis showed that among various mouse organs including brain, heart, kidney, liver, lung, muscle, skin, testis, spleen, and bladder, expression of Bre1b was highest in testis and spleen (Fig. 5A), suggesting that the Bre1a/Bre1b complex may play essential roles in these organs. We observed strong Bre1b staining in spermatogonia (Fig. 5B, left panel). We also found a strong signal for H3K79me2 and H3K4me3, the 2 modifications affected by ubiquitination of H2B. H3K79me2 was highest in middle meiosis (Fig. 5B), whereas H3K4me3 was strongest at the beginning and in the middle of meiosis (Fig. 5B), consistent with the roles these modifications play during yeast meiosis (34–36). Importantly, the staining for total histone H3 was uniform throughout the sections (Fig. 5B).

Loss of BRE1A/B is associated with development of seminoma. A, Bre1b is highly expressed in mouse testis. B, Bre1b levels are the highest in meiotic prophase cells (depicted by arrows). Staining of Bre1b, H3K79me2, H3K4me3, and of total histone H3 is shown. C, protein levels of BRE1B, H3K79me, and H3K4me3 are lower in seminoma compared with normal testis. D, H3K79me2 is significantly lower in seminoma compared with normal testis and nonseminomatous cancers of testis. E, representative pictures show that loss of Bre1A/B and H3K79me2 occur early in seminoma development. Normal testis, CIS–carcinoma in situ, and seminoma sections are taken from same patients. Arrows point to CIS. Asterisks denote a significant difference from normal tissue section (***, P < 0.001; **, P < 0.01, t test).

Having shown the importance of Bre1 homologs to maintenance of genomic stability, we hypothesized that suboptimal expression of Bre1 homologs in testis might be associated with testicular cancer. To test this supposition, we analyzed BRE1A (RNF20) and BRE1B (RNF40) expression data available from public databases (37, 38) and found significantly lower levels of BRE1A mRNA in human seminoma compared with normal testicular tissue (Supplementary Fig. S6). Seminoma is a type of testicular germ cell cancer that is the most common solid tumor in otherwise healthy men aged 15 to 35 years. It is widely accepted that seminoma arises from the precursor lesion carcinoma in situ (CIS, also known as intratubular germ cell neoplasia) and can grow rapidly and metastasize (39). Seminomas are characterized by high levels of genome instability and gains of chromosome 12p, which are not present in CIS. Although formerly often lethal, seminoma is highly sensitive to ionizing radiation therapy and chemotherapy and most are now cured.

To assess BRE1A/B protein levels, we stained tissue microarrays containing tissue sections from normal testis and seminoma. Levels of both BRE1B protein and BRE1A/B-dependent dimethylation of H3K79 and trimethylation of H3K4 were significantly lower in seminoma compared with normal tissue (Fig. 5C). Further analysis of publicly available gene expression arrays (37, 38) showed that among different testicular cancers seminoma displayed the lowest levels of BRE1A (Supplementary Fig. S6), and by staining the testis tissue microarrays, we found that among testicular cancers, H3K79me2 was also lowest in seminoma (Fig. 5D, Supplementary Fig. S7 and Supplementary Table S4). Importantly, staining of BRE1A and H3K79me2 in CIS was also lower than in normal tissue from the same patients (Fig. 5E), suggesting that deficiency in BRE1A/B and in methylation of H3K4 and H3K79 occurs before acquisition of an invasive phenotype.

Discussion

We show that Bre1 (human BRE1A/B (RNF20/40) and mouse Bre1a/b (Rnf20/40)) acts as an important suppressor of chromosomal instability (CIN). This finding complements the previously suggested mechanism for Bre1 tumor suppression through transcriptional regulation of select oncogenes and tumor suppressor genes (14). The types of chromosomal aberrations we observed after knockdown of Bre1 indicated that a defect in HR contributes to CIN in the Bre1-deficient cells (40, 41). This conclusion is consistent with our previous observation that reduced monoubiquitination of H2B in Saccharomyces bre1null mutants and in mouse cells leads to defective recombinational repair of DSBs; refs. 10, 42). We show that R-loops, the RNA–DNA hybrid structures usually formed behind elongating RNA polymerase II when mRNA processing is disturbed (30–32), constitute a significant source of DSBs in Bre1-deficient cells. Overall, our data support a model in which reduction in Bre1-dependent ubiquitination of histone H2B increases genomic instability through increased generation of DSBs resulting from a defect in correct processing of canonical histone mRNA and through inhibition of HR needed for DSB resolution (Fig. 6).

A model depicting sources of genomic instability in Bre1-deficient cells. Loss of Bre1 leads to increase in DSBs due to defects in homologous recombination and in the processing of canonical histone mRNAs. Incorrect processing of replication-dependent histone mRNA 3′-ends facilitates cotranscriptional formation of R-loops, which block replication forks and result in DSB.

It should be noted that although we interpret the observed Bre1 knockdown phenotype as arising from an impact on the well-known role of Bre1 in H2B ubiquitination, it is formally possible that additional targets for the Bre1 ubiquitin ligase exist, which could contribute to the knockdown phenotype. Testing this with an H2B K120 substitution mutant is not straightforward in mammals because their genomes contain at least 17 H2B genes (43). However, studies using overexpressed ectopic H2BK120R mutant gene in cells with wild-type chromosomal copies of H2B have shown that effects of overexpression of H2BK120R mimic those of loss of BRE1, including the effect on the formation of γH2AX foci, which we used in our study to detect DSBs (11, 12). Also, in Saccharomyces, in which chromosomal copies of the H2B gene number only 2 and can be deleted, H2BK123R mutants do mimic the bre1 deletion phenotype (reviewed in ref. 9), implying that the phenotypes are likely to be conferred through effects on the H2B target. Questions remain, however, as to whether the phenotypes we observed after loss of Bre1 are mediated via the well-known impact of H2B ubiquitination on methylation of histone H3, and, specifically, whether H3K4me3 and or H3K79me2 are involved. Detailed investigation of the role that H3K4 methylation plays in different Bre1 phenotypes is hampered by the fact that there are many SET1 homologs in mammals, some of which may not be uH2B-dependent (reviewed in ref. 9). Also, knocking down the H3K79 methyltransferase Dot1 would completely eliminate methylation of H3K79, whereas loss of Bre1-mediated H2B ubiquitination only eliminates the higher states of methylation of H3K79, so additional phenotypes may be conferred by the Dot1 knockdown beyond those mediated by the impact of uH2B on H3K79. Although the effects of Dot1 knockdown in mice (44, 45) paralleled the impaired cell growth, increased ploidy and centromeric abnormalities, we observed that after Bre1 knockdown, specific clarification of these issues requires further study.

A crucial unresolved question in cancer biology is whether CIN represents an early event and is therefore a driving force of carcinogenesis. Our results support models in which CIN drives tumorigenesis rather than being its consequence. We provide a comprehensive demonstration of a stepwise accumulation of chromosomal changes that start with downregulation of Bre1. Consistent with the CIN model, we also show that BRE1A/B deficiency accompanies early steps of testicular cancer development.

Correct processing of replication-associated histone mRNA is particularly relevant in connection to testicular carcinogenesis. In the testis, a massive synthesis of histone variants accompanies dramatic reorganization of the genome, during which the majority of the histones are replaced by transition proteins and protamines. Lack of Bre1 leading to abnormal presence of polyadenylated histone mRNAs, which are not rapidly degraded at the end of S-phase, could interfere with proper incorporation of the variant histones into chromatin, and lead to testicular dysgenesis. We showed that low levels of BRE1A and BRE1B, and low H3K79me2 are found in intratubular cell neoplasia (CIS), as well as in seminomas. Seminomas show chromosomal changes similar to those found in CIS and therefore are considered a default pathway from the CIS precursor lesion to invasive testicular germ cell tumors (TGCT; ref. 39). Like all TGCTs, seminomas are characterized by high levels of CIN and aneuploidy, and a gain of chromosome 12p (46). Gain of 12p is not present in CIS, and so it is believed that overexpression of gene(s) on 12p is pertinent to invasive growth. Thus, we can speculate that genomic instability initiated by the abnormal downregulation of BRE1A/BRE1B function may facilitate gain of 12p and thus constitute one possible route to seminoma. Seminomas recapitulate the undifferentiated and pluripotent primordial germ cell (PGC) phenotype and are thought to arise when a block in maturation of PGCs prevents them from forming spermatogonia. Downregulation of BRE1A/BRE1B may be associated with such a maturation block (39) and thus may lead to infertility. In fact, men from families with fertility problems are known to have an elevated risk of testicular cancers, especially seminoma (47–49). Hence, deficiency in BRE1A/B may be among etiologic factors in common for both infertility and testicular cancer. In addition, mutation of BRE1 (RNF20) has been found among other CIN genes mutated in colorectal cancers (50) suggestive of a more general role of Bre1 in CIN.

In conclusion, we propose that the mammalian homologs of the yeast BRE1 gene serve as tumor suppressors by preventing replication stress and chromosomal instability that arise from DSBs associated with incorrect processing of replication-associated histone mRNAs and inefficient HR. In addition to clarifying basic cellular mechanisms, the identification of Bre1 as a CIN gene may have specific relevance for estimation of risk and diagnosis of testicular cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

The work was supported by grant CA67166 awarded to JMB by the National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis.
Cytometry A2006;69:212–21.